Small cell lung cancer (SCLC) causes over 200,000 patient deaths worldwide each year. A major factor contributing to the dismal outcome of SCLC, as in many solid tumors, is resistance to current therapies that have remained largely the same for the past 30 years. Cell-of-origin and molecular determinants of SCLC recurrence as well as progression are fascinating yet poorly understood subjects of immense clinical importance. Despite evidence of pre-malignant cells possessing the same tumor-initiating mutations and potential implications in tumor progression, characterization of these cells has been challenging due to lack of tractable model and informed ways of defining aberrant processes. Using genetically engineered mouse models of SCLC, we isolated pre-malignant cells with specific lineage-tracer and established lines of the cells that lack known oncogenic aberrations and do not undergo spontaneous transformation in culture and allograft model. Comparative molecular profiling of the pre-malignant cell and SCLC cell resulted in identification of `drivers' pathologically relevant to tumor progression as opposed to ones identified in conventional approaches comparing whole lung and tumor. The objectives of this proposal are to define molecular properties of preSC as well as its malignant progression using integrative gene expression analyses and functional genetics, and to assess preSC as therapeutic target. The hypothesis is that plasticity in cholesterol metabolism defines pre- malignant precursor cells of SCLC (preSC) during tumor progression. This hypothesis has been formulated based on our preliminary findings pointing to increased cholesterol metabolism during malignant progression and the reduced growth of oncogene-transformed cells in response to a drug with anti-cholesterol properties. We propose the following specific aims. In Aim 1, we will determine roles of genes for cholesterol metabolism and its related signaling pathways, using virus-mediated gene expression approaches in vivo and in vitro. In Aim 2, we will test whether changes in systemic level of cholesterol metabolism influences tumor progression by modulating body cholesterol level using high-cholesterol diet or drugs known to regulate cholesterol synthesis and transport. In Aim 3, we will examine human lung tumors for deregulated cholesterol metabolism by analyzing proteins that play role in cholesterol intake and synthesis. To validate clinical significance of cholesterol metabolism in tumor progression, we aim to establish human pre-malignant cells and test effect of modulating cholesterol metabolism on oncogene-induced transformation of human pre-malignant cells. This proposal outlines a first step toward understanding detailed molecular mechanisms of key driver molecules during tumor progression, which will pave the way for even more exciting experiments aimed at developing novel strategies for prevention and treatment. Lastly, successful outcomes of this proposal could provide a paradigm of identifying key determinant of many cancers given that cell-of-origins and lineage markers for those caners will be known in the near future.